MODULAR TISSUE SCAFFOLDING TOOLS: A NEW FAMILY OF SELF-ASSEMBLED BIOMATERIALS DERIVED FROM COPPER-CAPILLARY ALGINATE GELS By BRADLEY JAY WILLENBERG A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2005 Copyright 2005 by Bradley Jay Willenberg To the old man who drowned so near the shore. ACKNOWLEDGMENTS I would like to sincerely thank Dr. Christopher Batich, my supervising committee chairman. His hands-off approach and wealth of scientific and engineering knowledge propelled me and this project faster and farther than initially envisioned. I thank Dr. Anthony Brennan, Dr. Robert DeHoff and Dr. Thomas Mareci for their time and efforts as my teachers and committee members. I also specially thank Dr. Naohiro Terada and Dr. Takashi Hamazaki for their tremendous efforts, input and profound support of this work. Much praise and thanks go to Marina Scotti for giving me roots; split a piece of wood and she is there. Lift a stone and you will find her. I thank my family for their consistent support, guidance, criticism and strength. I especially thank, Mom, Jimbo, Dan and Ryan, Dr. Amelia Dempere and Specialist Wayne Acree (“The Lab Dude”). I also thank the entire Major Analytical Instrumentation Center (MAIC) staff for their scientific and social insights. I further thank all those students, faculty and staff who took the time to know and talk with me. Special thanks go to Charlie Murphey (Precision Tool & Engineering, Gainesville, FL) for custom machining all my tools and reactors, and to Dr. Charles (Chuck) Seegert for shaping my early scientific and tissue-engineering thinking. iv TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................................................................................. iv LIST OF FIGURES ......................................................................................................... viii LIST OF OBJECTS .............................................................................................................x ABSTRACT....................................................................................................................... xi CHAPTER 1 INTRODUCTION ........................................................................................................1 2 BACKGROUND AND SIGNIFICANCE....................................................................5 3 MATERIALS AND METHODS ...............................................................................15 Scaffold Synthesis ......................................................................................................15 Raw Copper-Capillary Alginate Gel (RCCAG)..................................................15 Classic descending growth technique ..........................................................16 Time-lapse videoscopy.................................................................................17 Barium Stabilized Copper-Capillary Alginate Gel (BCCAG) ............................17 Exchange-reactor design and setup..............................................................17 Barium hydroxide processing.......................................................................18 Oligochitosan-Barium (OBCCAG) and Oligochitosan (OCCAG) Stabilized RCCAG............................................................................................................19 Scaffold Characterization ....................................................................................20 Optical microscopy ......................................................................................20 Scanning electron microscopy .....................................................................21 Percent Water Content Determination.................................................................22 Biological Assesment .................................................................................................22 In Vitro Study: Swiss Albino Embryonic Mouse Fibroblasts Expressing Green Fluorescent Protein (GFP-3T3).............................................................24 In Vitro Study: Mouse Embryonic Stem Cells Expressing Green Fluorescent Protein (GFP-mES)..........................................................................................24 Evaluation of mES cell growth, survival and morphology vs. time.............24 Comparison of ES maintenance (LIF+) and ES differentiation (LIF-) media conditions.....................................................................................25 v 4 RESULTS AND DISCUSSION.................................................................................26 Scaffold Synthesis and Processing .............................................................................26 RCCAG ...............................................................................................................26 Growth videos ..............................................................................................27 Growth kinetics ............................................................................................27 Storage concerns ..........................................................................................28 BCCAG ...............................................................................................................29 Colorimetric changes during barium hydroxide treatment ..........................29 Exchange-reactor advantages and difficulties..............................................30 OBCCAG and OCCAG.......................................................................................31 Consequences of Media Wash.............................................................................32 Scaffold Characterization ...........................................................................................32 Optical Microscopy .............................................................................................33 RCCAG ........................................................................................................33 Evidence of precipitates within BCCAG .....................................................34 Scanning Electron Microscopy/Energy Dispersive Spectroscopy (SEM/EDS) and X-ray Mapping..........................................................................................35 Consequences of freeze-drying ....................................................................35 RCCAG Data................................................................................................35 BCCAG Data................................................................................................37 OCCAG Data ...............................................................................................39 Summary of morphologic and compositional analysis ................................39 Equilibrium Water Weight Percent Analysis ......................................................40 Biologic Assessment...................................................................................................41 Mouse Embryonic Fibroblasts (GFP-3T3)..........................................................41 Mouse Embryonic Stem Cells (GFP-mES).........................................................42 Evaluation of cell Growth, survival and morphology vs. time ....................43 Confocal microscopy and video data ...........................................................43 Comparison of ES maintenance (Lif+) and ES differentiation (Lif-) media conditions................................................................................................44 5 CONCLUSIONS ........................................................................................................62 Introduction.................................................................................................................62 Scaffold Synthesis: The Agony and the Ecstasy ........................................................62 Characterization: CCAG Scaffolds as Subtle Composites .........................................63 Biological Assessment: Living with Success and Failure ..........................................64 6 FUTURE WORK........................................................................................................66 Introduction.................................................................................................................66 Synthesis: Expanding the CCAG Scaffold Family.....................................................66 Scaffold Characterization: Quantitative Bulk and Surface Compositional Analysis .66 Biologic Assessment: Standardized Methods and Controls for Stem Cell Studies....67 APPENDIX PUBLICATIONS, PRESENTATIONS AND PATENTS.........................69 vi LIST OF REFERENCES...................................................................................................70 BIOGRAPHICAL SKETCH .............................................................................................76 vii LIST OF FIGURES Figure page 2-1 Peripheral nerve hierarchical structure.....................................................................13 2-2 Molecular structures of alginate and oligochitosan polymers..................................14 3-1 Raw-CCAG (RCCAG) classical descending technique synthesis scheme..............17 3-2 Teflon™ exchange-reactor.......................................................................................18 4-1 Plot of RCCAG growth as a function of time.